Human immunodeficiency virus (HIV) is a rapidly evolving pathogen that escapes immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies (bnAbs) by vaccination require a deeper understanding of evolution of the immune response to infection, since these protective antibodies typically take ~4-5 years to develop. In HIV infected individuals, viruses and antibody producing B-cells evolve together, creating a virus-antibody ?arms race?, with populations of viruses and antibodies present throughout infection. The proposed research is to analyze critical early time-points of the arms race in a donor who developed antibodies of significant breadth, to guide immunogen design. In addition to rapid mutation, HIV also uses heavy glycosylation and conformational masking to evade the immune system. Donor CH848 produced a bnAb lineage, called DH270, which interacts with the glycan ?supersite? at the base of the HIV envelope (Env) variable loop V3. Analysis of crystal structures of complexes between mature members and fragments of the HIV Env, together with binding data, suggest that improbable mutations in the antibodies led to the different neutralization properties of antibodies in the different branches of the lineage, without any major structural change in the antibody paratope or antigen epitope. While many V3-glycan ?supersite? bnAbs recognize the N332 glycan, their actual epitopes differ in other glycans and Env peptides they recognize. Thus, it remains to be determined what triggered DH270 lineage development. To understand properties of HIV Env and interactions with antibodies that were critical for DH270 lineage development, atomic resolution structures of HIV Envs will be determined by cryo-electron microscopy and/or X-ray crystallography with an early member of the DH270 lineage, DH270.IA4, and with cooperating antibody lineage members, DH475 and DH0022. Cooperating antibodies, also produced in the CH848 donor, triggered virus escape mutations that improved binding to DH270 lineage antibodies and likely accelerated affinity maturation in the DH270 lineage. Hypotheses on how the DH270 lineage progenitor antibody could bind Env may also be deduced from the DH270.IA4 complex structure, since DH270.IA4 differs from the progenitor by five amino acids. Hypotheses will be tested by introducing mutations into the Fabs and/or HIV Env and determining binding affinities by biolayer interferometry. Structures of cooperating antibodies in complex with Env will identify properties of HIV Env (i.e., conformation, glycosylation patterns, etc.) that triggered these non- neutralizing antibodies, and despite their overlapping epitopes, how they aided DH270 lineage development. These data will identify mechanism(s) that triggered the development of broadly neutralizing glycan- dependent antibodies, and guide vaccine design. Undergraduate research students supported by this grant proposal will explore an issue of critical public health importance using cutting edge techniques, be co-authors on published work and be mentored by experts committed to their long-term career development.